Cytokinins are mitogenic plant hormones that control multiple aspects of growth and development, including regulation of cell division and metabolism, stimulation of chloroplast, developmen, modulation of shoot and root development, and delay of leaf senescence (Hwang, et al. (2012) Annu. Rev. Plant Biol. 63:353-380; Perilli, et al. (2010) Curr. Opin. Plant Biol. 13(1):21-26). Cytokinin signals are transmitted through a multistep histidine-to-aspartate phosphorelay system, evolutionarily related to the two-component signaling systems of prokaryotes (Hwang, et al. (2012) supra; Schaller, et al. (2011) Curr. Biol. 21(9):R320-330). In Arabidopsis, cytokinins are perceived by the three receptors AHK2 (for ARABIDOPSIS HISTIDINE KINASE 2), AHK3, and AHK4, which, upon perception of the cytokinin signal, autophosphorylate a conserved His residue (Inoue, et al. (2001) Nature 409(6823):1060-1063; Ueguchi, et al. (2001) Plant Cell Physiol. 42(7):751-755). The regulatory phosphoryl group is passed from receptor to a histidine-containing phosphotransfer (AHP) protein, and from there to a type-B response regulator (ARR). Phosphorylation of the Arabidopsis type-B ARR proteins modulates their ability to control gene expression as the key transcription factors in the primary response pathway (Kim, et al. (2006) Proc. Natl. Acad. Sci. USA 103(3):814-819; Argyros, et al. (2008) supra). Among the transcriptional targets of the type-B ARRs are a second class of response regulators, the type-A ARRs, which act as negative regulators of the signal transduction pathway (To, et al. (2004) Plant Cell 16(3):658-671). This model of cytokinin signal transduction has been established largely based on studies in Arabidopsis, but similar two-component signaling components have been identified in other plant species such as the monocot rice and the moss Physcomitrella patens, supporting a common pathway for the transmission of the cytokinin signal in land plants (Pils & Heyl (2009) Plant Physiol. 151(2):782-791; Tsai, et al. (2012) Plant Physiol. 158(4):1666-1684).
Control of protein stability through ubiquitin-mediated proteolysis has emerged as a central theme in plant growth and development over the past decade (Hua & Vierstra (2011) Annu. Rev. Plant Biol. 62:299-334). E3 ubiquitin ligases have been identified that target key signaling elements for degradation in many plant hormone signaling pathways, including those for auxin, ethylene, gibberellin, and jasmonic acid (Santner & Estelle M (2010) Plant J. 61(6):1029-1040). Identification of such a regulatory component for cytokinin signal transduction has not been confirmed, though several studies suggest that the ubiquitin-proteasome system plays a role in the regulation of cytokinin signaling (Kim, et al. (2012) Plant J. 69(6):934-945; Dharmasiri, et al. (2005) Nature 435(7041):441-445; Zheng, et al. (2011) Plant Physiol. 156(4):1878-1893; Smalle, et al. (2002) Plant Cell 14(1):17-32; To, et al. (2007) Plant Cell 19(12):3901-3914). Most significantly, the stability of the type-B response regulator ARR2 decreases in the presence of cytokinin, and a more stable mutant version of ARR2 enhances cytokinin sensitivity in various developmental processes (Kim, et al. (2012) supra).
The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels (Houba-Herin (1999) Plant J. 17:615-626; Morris, et al. (1999) Biochem. Biophys. Res. Commun. 255:328-333; Brugiere, et al. (2003) Plant Physiol. 132:1228-1240). The catabolic enzyme cytokinin oxidase (CKX) plays a major role in controlling cytokinin levels in plant tissues and methods for modulating plant development via CKX have been described (U.S. Pat. No. 8,222,483). F-box proteins have also been suggested as targets for regulating cytokinin production See US 2012/0216318. However, the use of a particular F-box from the hundreds of F-box proteins identified in plants has not been suggested.
In view of the influence of cytokinins on a wide variety of plant developmental processes, including root architecture, shoot and leaf development, and seed set, the ability to manipulate responses to cytokinin in higher plant cells, and thereby affect plant growth and productivity, is of great commercial value.